Morphology, biophysical properties and protein-mediated fusion of archaeosomes

As variance from standard phospholipids of eubacteria and eukaryotes, archaebacterial diether phospholipids contain branched alcohol chains (phytanol) linked to glycerol exclusively with ether bonds. Giant vesicles (GVs) constituted of different species of archaebacterial diether phospholipids and glycolipids (archaeosomes) were prepared by electroformation and observed under a phase contrast and/or fluorescence microscope. Archaebacterial lipids and different mixtures of archaebacterial and standard lipids formed GVs which were analysed for size, yield and ability to adhere to each other due to the mediating effects of certain plasma proteins. GVs constituted of different proportions of archaeal or standard phosphatidylcholine were compared. In nonarchaebacterial GVs (in form of multilamellar lipid vesicles, MLVs) the main transition was detected at T_m = 34. 2°C with an enthalpy of ΔH = 0.68 kcal/mol, whereas in archaebacterial GVs (MLVs) we did not observe the main phase transition in the range between 10 and 70°C. GVs constituted of archaebacterial lipids were subject to attractive interaction mediated by beta 2 glycoprotein I and by heparin. The adhesion constant of beta 2 glycoprotein I – mediated adhesion determined from adhesion angle between adhered GVs was in the range of 10⁻⁸ J/m². In the course of protein mediated adhesion, lateral segregation of the membrane components and presence of thin tubular membranous structures were observed. The ability of archaebacterial diether lipids to combine with standard lipids in bilayers and their compatibility with adhesion-mediating molecules offer further evidence that archaebacterial lipids are appropriate for the design of drug carriers.     

A Fourier transform infrared investigation of the structural differences between ribonuclease A and ribonuclease S

Fourier transform infrared spectroscopy was used to investigate the small conformational differences which exist between ribonuclease A and ribonuclease S in aqueous systems. Deconvolution and derivative methods were used to observe the overlapping components of the amide I and II bands. These proteins give identical spectra in H2O and after complete exchange in 2H2O. However structural differences are revealed by monitoring the rate of 1H-2H exchange by Fourier transform infrared spectroscopy. At equivalent times of exposure in 2H2O buffer ribonuclease S undergoes greater isotopic exchange than ribonuclease A. Thus complete exchange takes place for ribonuclease S but not ribonuclease A after incubation at room temperature for 8 days. Complete 1H-2H exchange of ribonuclease A was achieved by incubation at 62 degrees C for 30 min. The available X-ray data and comparison with the infrared spectra of other soluble proteins was used to assign the components of the amide I and II bands to various secondary structures. In particular, band shifts observed during the later stages of exchange are associated with slowly exchanging residues in beta-strand and alpha-helical regions. The higher rate of exchange for ribonuclease S is associated with a greater conformational flexibility and a more open structure. The results show that it is necessary to be cautious in making band assignments based on exchange methods unless the extent of exchange is known. Furthermore, it is seen that the combination of Fourier transform infrared spectroscopy and hydrogen-deuterium exchange is a powerful technique for revealing small differences in protein secondary structure.     

Separation of hemoglobin types by cation-exchange high-performance liquid chromatography

The use of a recently developed cation-exchange HPLC packing material for the separation of hemoglobin types in human blood has been investigated. Adult and newborn hemolysates from normal individuals and from subjects with hemoglobin disorders were analyzed using a weak cation carboxymethyl-bonded phase on 5-micron-particle-size silica. Elution was accomplished using a Bistris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1, 3-propanediol) gradient. Seven well-resolved HbA1 fractions eluted before the major HbA peak. Hbs A1a, A1b, A1c and an HbA1 fraction that increased with aging of the hemolysates were separately eluted. HbF when present or when added to the hemolysates eluted as a distinct peak. HbA was followed by Hbs A2, S, and C when present. An early-eluting peak corresponding to Hb Bart's was identified in newborn hemolysates. It is concluded that cation-exchange HPLC provides a new tool for the reliable separation of minor hemoglobin components.     

Some biochemical properties of an acido-thermophilic archaebacterium,Sulfolobus acidocaldarius

To elucidate the phylogenic status of archaebacteria, some basic cellular components of an acido-thermophilic archaebacterium,Sulfolobus acidocaldarius, were studied. Poly(A) containing RNA was present in the cells, and performed the role of mRNA in a cell-free extract of reticulocyte or the archaebacteria. Poly(A) containing RNA was also found in other archaebacterial cells. The absence of cap structure was suggested in these RNAs. The cell-free protein synthesis using the archaebacterial extract was inhibited by anisomycin, a specific inhibitor for eukaryotic ribosomes. Two unique membrane-bound ATPases were detected. Based on resistance to H⁺-ATPase inhibitors, these enzymes seemed not to be F₀F₁-ATPase.     

Rapid screening of the fermentation profiles of wine yeasts by Fourier transform infrared spectroscopy

A rapid screening method for the evaluation of the major fermentation products of Saccharomyces wine yeasts was developed using Fourier transform infrared spectroscopy and principal component factor analysis. Calibration equations for the quantification of volatile acidity, glycerol, ethanol, reducing sugar and glucose concentrations in fermented Chenin blanc and synthetic musts were derived from the Fourier transform infrared spectra of small-scale fermentations. The accuracy of quantification of volatile acidity in both Chenin blanc and synthetic must was excellent, and the standard error of prediction was 0.07 g l(-1) and 0.08 g l(-1), respectively. The respective standard error of prediction in Chenin blanc and synthetic musts for ethanol was 0.32% v/v and 0.31% v/v, for glycerol was 0.38 g l(-1) and 0.32 g l(-1), for reducing sugar in Chenin blanc must was 0.56 g l(-1) and for glucose in synthetic must was 0.39 g l(-1). These values were in agreement with the accuracy obtained by the respective reference methods used for the quantification of the components. The screening method was applied to quantify the fermentation products of glycerol-overproducing hybrid yeasts and commercial wine yeasts. Principal component factor analysis of the fermentation data facilitated an overall comparison of the fermentation profiles (in terms of the components tested) of the strains. The potential of Fourier transform infrared spectroscopy as a tool to rapidly screen the fermentative properties of wine yeasts and to speed up the evaluation processes in the initial stages of yeast strain development programs is shown.     

Assay of tryptophan hydroxylase and aromatic L-amino acid decarboxylase based on rapid separation of the reaction product by high performance liquid chromatography

A rapid separation of 5-hydroxytryptophan by high performance liquid chromatography (HPLC) was achieved for the assay of tryptophan hydroxylase. "Bulk separation" of the product from all other components in the reaction mixture by HPLC was achieved by 1) the choice of a suitable column-solvent system so as to elute the reaction product ahead of other components in the sample mixture, 2) the use of a monitor selective for the reaction product, 3) minimization of the column length so as to achieve rapid separation of the product from the substrate. The method finally employed a reversed phase column of 5 cm length, relatively rapid elution at 2 ml/min and fluorescence detection at 350 nm with an excitation at 302 nm. The assay is convenient and as sensitive as the radioisotope method. The advantages of the method are 1) almost no pretreatment of samples, 2) repeatability every 2 min, 3) wide latitude of product determination from picomole to nanomole amounts per assay. The method was extended to the assay of 5-hydroxytryptophan decarboxylase by essentially the same procedures.     

Development of a quantification system of ionic dissociative metabolites using an FT-IR/ATR method

A simultaneous quantification system of ionic dissociative metabolites was developed using a Fourier transform mid-infrared spectroscopic method by focusing our attention on the enzyme reaction from glucose 6-phosphate to fructose 6-phosphate with phosphoglucose isomerase (PGI). We studied the pH dependency of the infrared spectra of the mixture solution for which the PGI reaction was assumed. The infrared spectra of ionic dissociative components in the mixture solution were extracted by multiple linear regression analysis under the assumption of ionic dissociation equilibrium. Additionally, we constructed a simultaneous quantification system using the extracted spectra of the ionic dissociative components on the basis of the ionic dissociation equilibrium. We could accurately estimate the pH value and the concentrations of the ionic dissociative materials in their mixture solution by using this quantification system. In addition, the stability of quantification results for a pK shift was also verified.     

Chemical composition of acid–base fractions separated from biooil derived by fast pyrolysis of chicken manure

Our earlier investigations on the chemical composition of biooils derived by the fast pyrolysis of chicken manure revealed the presence of more than 500 compounds. In order to simplify this heterogeneous and complex chemical system, we produced four biooil fractions namely strongly acidic fraction A, weakly acidic fraction B, basic fraction C and neutral fraction D on the basis of their solubilities in aqueous solutions at different pHs. The yield (wt/wt.%) for fraction A was 3%, for fraction B 21.3%, for fraction C 2.4% and for fraction D 32.4%, respectively. The four fractions were analyzed by elemental analyses, Fourier Transform infrared spectrophotometry (FTIR), ¹H and ¹³C nuclear magnetic spectroscopy (NMR), and electrospray ionization mass spectrometry (ESI-MS). The major components of the four fractions were saturated and unsaturated fatty acids, N-heterocyclics, phenols, sterols, diols and alkylbenzenes. The pH separation system produced fractions of enhanced chemical homogeneity.     

Electrochemical and ultraviolet/visible/infrared spectroscopic analysis of heme a and a3 redox reactions in the cytochrome c oxidase from Paracoccus denitrificans: separation of heme a and a3 contributions and assignment of vibrational modes

Cytochrome c oxidase from Paracoccus denitrificans was studied with a combined electrochemical and ultraviolet/visible/infrared (UV/vis/IR) spectroscopic approach. Global fit analysis of oxidative electrochemical redox titrations was used to separate the spectral contributions coupled to heme a and a3 redox transitions, respectively. Simultaneous adjustment of the midpoint potentials and of the amplitudes for a user-defined number of redox components (here heme a and a3) at all wavelengths in the UV/vis (900-400 nm) and at all wavenumbers in the infrared (1800-1250 cm-1) yielded difference spectra for the number of redox potentials selected. With an assumption of two redox components, two spectra for the redox potential at -0.03 +/- 0.01 V and 0.22 +/- 0.04 V (quoted vs Ag/AgCl) were obtained. The method used here allows the separation of the heme signals from the electrochemically induced visible difference spectra of native cytochrome c oxidase without the addition of any inhibitors. The separated heme a and a3 UV/vis difference spectra essentially correspond to spectra obtained for high/low-spin and 5/6-coordinated heme a/a3 model compounds presented by Babcock [(1988) in Biological Applications of Resonance Raman Spectroscopy (Spiro, T., Ed.) Wiley and Sons, New York]. Single-component Fourier transform infrared (FTIR) difference spectra were calculated for both hemes on the basis of these fits, thus revealing contributions from the reorganization of the polypeptide backbone, from the hemes, and from single amino acids upon electron transfer of the cofactors (heme a/a3, CuA, and CuB), as well from coupled processes such as proton transfer. A tentative assignment of heme vibrational modes is presented and the assignment of the signals to the reorganization of the polypeptide backbone and to perturbations of single amino acids, in particular Asp, Glu, Arg, or Tyr, is discussed.     

Isolation of peptide antibiotic virginiamycin components and selection of their producer Streptomyces virginiae]

A method for chromatographic separation and quantitative determination of individual components of the antibiotic virginiamycin, produced by microbiological synthesis (Streptomyces virginiae strain 147), is described. The components, M1-2 and S1-5, were isolated from fermentation broth and identified by HPTLC and HPLC (the results obtained using the two methods correlate well with each other). Conditions of culturing of the producer and compositions of nutritive media were optimized. Using UV irradiation as a mutagenic factor, the producer was selected for increased level of synthesis of the antibiotic; this was achieved by inducing mutations that impart resistance to virginiamycin and meta-fluorophenylalanine, an analog of phenylalanine.     

The detection of radical scavenging compounds in crude extract of borage (Borago officinalis L.) by using an on-line HPLC-DPPH method

The rapid evaluation of antioxidant activity of crude borage (Borago officinalis L.) extract was determined by using DPPH free radical method. This borage extract resulted in a rapid decrease of the absorbance and showed very high hydrogen-donating capacity towards the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical. A new HPLC-DPPH on-line method was applied for a screening of several radical scavenging components in this borage extract as well as for quantitative analysis. This on-line HPLC-DPPH method was developed using a methanolic solution of DPPH-stable radical. The HPLC-separated analytes reacted post-column with the DPPH solution in methanol. The induced bleaching was detected as a negative peak photometrically at 515 nm. The separation of antioxidative components was carried out by gradient HPLC with mobile-phase composition ranging from 2% to 80% acetonitrile with 2% acetic acid in water, UV detection was carried out at 280 nm. The HPLC analysis of borage extract revealed the presence of several radical scavenging components in the borage extract. The results obtained from the chromatograms suggest that some compounds present in the extract possess high radical quenching ability. The dominant antioxidative compound in the crude extract of borage leaves was identified as rosmarinic acid.     

Quantitative high-performance liquid chromatography of nucleosides and bases in human plasma

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the estimation of purines, pyrimidines and their congeners in biological fluids. An initial HPLC separation allowed the collection of a number of effluent fractions, each of which contained a single component of interest. The re-application of these fractions to a second HPLC separation permitted the resolution and quantification of nanogram amounts of these components. Isocratic elution with volatile buffers renders the samples amenable to automatic sampling procedures or lyophilisation. Data are presented on the application of the method to the analysis of nucleosides and bases in human plasma.     

The binding site of ribosomal protein L10 in eubacteria and archaebacteria is conserved: reconstitution of chimeric 50S subunits.

It has been shown by electron microscopy that the selective removal of the stalk from 50S ribosomal subunits of two representative archaebacteria, namely Methanococcus vaniellii and Sulfolobus solfataricus, is accompanied by loss of the archaebacterial L10 and L12 proteins. The stalk was reformed if archaebacterial core particles were reconstituted with their corresponding split proteins. Next, structurally intact chimeric 50S subunits have been reconstituted in vitro by addition of Escherichia coli ribosomal proteins L10 and L7/L12 to 50S core particles from M vaniellii or S solfataricus, respectively. In the reverse experiment, using core particles from E coli and split proteins from M vaniellii, stalk-bearing 50S particles were also obtained. Analysis of the reconstituted 50S subunits by immunoblotting revealed that E coli L10 was incorporated into archaebacterial core particles in both presence or absence of E coli L7/L12. In contrast, incorporation of E coli L7/L12 into archaebacterial cores was only possible in the presence of E coli L10. Our results suggest that in archaebacteria - as in E coli - the stalk is formed by archaebacterial L12 proteins that bind to the ribosome via L10. The structural equivalence of eubacterial and archaebacterial L10 and L12 proteins has thus for the first time been established. The chimeric reconstitution experiments provide evidence that the domain of protein L10 that interacts with the ribosomal particle is highly conserved between eubacteria and archaebacteria.     

Plant fructans stabilize phosphatidylcholine liposomes during freeze-drying.

Fructans have been implicated as protective agents in the drought and freezing tolerance of many plant species. A direct proof of their ability to stabilize biological structures under stress conditions, however, is still lacking. Here we show that inulins (linear fructose polymers) isolated from chicory roots and dahlia tubers stabilize egg phosphatidylcholine large unilamellar vesicles during freeze-drying, while another polysaccharide, hydroxyethyl starch, was completely ineffective. Liposome stability was assessed after rehydration by measuring retention of the soluble fluorescent dye carboxyfluorescein and bilayer fusion. Inulin was an especially effective stabilizer in combination with glucose. Analysis by HPLC showed that the commercial inulin preparations used in our study contained no low molecular mass sugars that could be responsible for the observed stabilizing effect of the fructans. Fourier transform infrared spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry egg PtdCho by more than 20 degrees C in the presence of inulin. A direct interaction of inulin with the phospholipid in the dry state was also indicated by dramatic differences in the phosphate asymmetric stretch region of the infrared spectrum between samples with and without the polysaccharide.     

Archaebacterial ether lipid diversity analyzed by supercritical fluid chromatography: integration with a bacterial lipid protocol

A strategy has been developed for archaebacterial lipid analysis which provides three times the information to describe archaebacterial isolates and is compatible with simultaneous eubacterial/eukaryotic lipid analysis of environmental samples. Eubacterial and micro-eukaryotic biomass, community structure, and nutritional status have been routinely defined in environmental samples by lipid analysis. Lipid profiles are also useful in eubacterial identification and taxonomy. Polar lipid or whole cell ester-linked fatty acids are generally analyzed by gas chromatography-mass spectroscopy. Archaebacteria are characterized by their ether-linked membrane lipids. There is, however, less diversity in the side chains of archaebacterial membrane lipids as compared the eubacterial ester-linked membrane lipids. The information content of the archaebacterial lipid profile was increased by separately analyzing the polar lipid, glycolipid, and lipid-extracted residue fractions. Identification and quantification were performed by supercritical fluid chromatography. Results are presented for three species of methanogens and four thermoacidophile isolates, and compared with a literature review.     

Structural studies of phosvitin in solution and in the solid state

Phosvitin, a highly phosphorylated glycoprotein, represents the major fraction of hen egg yolk phosphoproteins. Circular dichroism, Fourier transform infrared spectroscopy, and Fourier transform infrared photoacoustic and fluorescence spectroscopic methods were employed to determine the secondary structure of the protein in both the solid and solution phases. This was supplemented by a Chou-Fasman type of predictive algorithm for the first 25 residues at the N terminus of the dephosphorylated protein. A three-compartment model consisting of alpha-helical, beta-sheet, and beta-turn components with beta-turns occurring at the interface between alpha-helical and beta-sheet regions in the proximity of O-phosphoserine residues is suggested from the combined analyses. Beta-sheets appear to be the dominant secondary structural component in phosvitin in the solid and solution phases. The suggested model bears many similarities to other phosphoproteins reported in the literature. The secondary structure of phosvitin is observed to be sensitive to environmental factors as previously reported although the present studies differ in some respects from earlier results. Preliminary results suggest that Ca2+ ions trigger a decrease in beta-sheet structure at pH 2.     

A simple procedure for the preparation and purification of the oligosaccharide components of the glycosylphosphatidylinositol anchor of membrane proteins

The oligosaccharide components of the glycosylphosphatidylinositol anchors of Trypanosoma brucei variant surface glycoproteins have been prepared and purified by treatment with hydrolytic enzymes and solvent extraction procedures followed by HPLC purification using a specific oligosaccharide binding matrix (Glyco-Pak N, by Waters). Three oligosaccharide peaks (peaks I, II and III) were resolved by a single isocratic HPLC step (70% acetonitrile in water). The material from these peaks was hydrolyzed in acid and analyzed by GC/MS. GC/MS analysis of the material obtained from each peak demonstrated the presence of inositol, glucosamine, and mannose in a 1:1:3 ratio. A variable number of galactose residues were detected in each peak. The galactose:inositol ratios of the purified components were 1:1, 2:1, and 3:1 for peaks I, II and III, respectively, suggesting that the separation obtained depends primarily on the number of sugar residues present in each fraction.     

An Improved Fluorometric High-Performance Liquid Chromatography Method for Sialic Acid Determination: An Internal Standard Method and Its Application to Sialic Acid Analysis of Human Apolipoprotein E

An improved fluorometric HPLC method for sialic acid determination was developed by employing synthetic N-propionylneuraminic acid (NPNA) as an internal standard. A fixed amount of NPNA was added to a sialoglycoconjugate sample. After hydrolyzing sialioglycoconjugates with diluted sulfuric acid, the released sialic acids and NPNA were derivatized with a fluorogenic compound, 1,2-diamino-4,5-(methylenedioxy)benzene (DMB), followed by fluorometric HPLC. The fluorescent derivative of NPNA was separated from those of N-acetylneuraminic acid, N-glycolylneuraminic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nonoic acid, and 2-keto-3-deoxyoctanoate on HPLC. The separation of NPNA derivative on HPLC was not interfered by components of biological samples such as human sera. Using this internal standard method, low amounts of NANA (0.15-1.0 ng) were quantified with the coefficient of variation values below 4%. Using this method, the sialic acid content of human apolipoprotein E was successfully determined. The present method is useful for sensitive and accurate quantification of sialic acids of different molecular species in biological samples.     

The primary structure of the hemoglobins of the adult jaguar (Panthera onco, Carnivora)

The primary structure of the hemoglobins from Jaguar (Panthera onco) are presented. Electrophoretic separations without and with a dissociating agent revealed the presence of two hemoglobin components, alpha 2 beta I2 and alpha 2 beta II2. The separation of the hemoglobin components was achieved by ion-exchange chromatography. The globin chains were separated by ion-exchange chromatography and also by reversed phase HPLC. The amino-acid sequences of the native chains and peptides were determined by liquid-phase and gas-phase sequencing. N-Acetylserine was detected by FAB-mass spectroscopy as N-terminal group of the beta I chain. The sequences are compared with that of human hemoglobin (Hb A).